Method for separating ionic species using capillary electrophoresis

ABSTRACT

A technique for separating, identifying and measuring ions in solution by capillary zone electrophoresis is described, which provides improved sensitivity and resolution of anionic and cationic species. The method involves introducing a sample containing the ionic species into a narrow core capillary filled with a carrier electrolyte containing a selected light-absorbing anion or cation to an electrical current in a capillary column causing the ions to elute according to their ionic mobility. Both UV absorbing and UV-transparent ions can be detected and quantitated by UV/Visible photometric monitoring.

RELATED APPLICATIONS

This application is a division of application Ser. No. 07/642,685, filedJan. 17, 1991, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/471,535, filed Jan. 29, 1990, entitled "MethodFor Separating Ionic Species Using Capillary Electrophoresis" by WilliamR. Jones, Petr Jandik and Michael Merion.

BACKGROUND

The separation and/or detection of ionic species is generally carriedout by utilizing electrochemical properties of analytes, such as ionicinteractions and conductivity in ion chromatography or ionic mobility incapillary electrophoresis. Ion chromatography (IC) is capable ofdetecting simultaneously a large variety of ionic species at lowconcentration levels. The ability to separate and detect several widelydifferent ionic species simultaneously is a unique characteristic of IC.In fact, the commercial viability of IC depends in part on its abilityto simultaneously separate and detect, inter alia, seven commoninorganic anions (F⁻, Cl⁻, NO₂ ⁻, Br⁻, NO₃ ⁻, HPO₄ ⁻ and SO₄ ⁻).However, there are important limitations to IC, including lack ofsufficient selectivity for certain types of mixtures, low separationefficiency and a relative complexity of instrumentation.

Capillary electrophoresis (CE) is an efficient analytical separationtechnique for analysis of minute amounts of sample. CE separations areperformed in a narrow diameter capillary tube, which is filled with anelectrically conductive medium termed the "carrier electrolyte". Acurrent is applied to the carrier electrolyte, and ionic species in thesample move from one electrode toward the other at a rate which isdependent upon certain characteristics, such as molecular charge, sizeand/or mobility. CE may be performed using gels or liquids, such asbuffers, in the capillary. In the liquid mode, known as free zoneelectrophoresis, separations are based on the ratio of charge to Stoke'sradius.

CE has several advantages over IC and conventional gel electrophoresisfor the separation of ionic species. These include improved resolutionand smaller sample size. In part, high resolution can be obtained sinceband broadening is minimized due to the narrow capillary diameter. Infree-zone electrophoresis, the phenomenon of electroosmosis, orelectroosmotic flow (EOF), which is the bulk flow of liquid rapidlymoves all of the sample molecules whether they are positively charged,negatively charged or neutral. Under certain conditions EOF cancontribute to improved separation speed in free-zone CE.

The detection of ionic species by CE is problematical particularly ifall seven of the common anions mentioned above are to be determinedsimultaneously. Most ions do not absorb light, so they cannot bedetected by conventional photometric means. e.g., direct photometric orfluorescent detection. However, these ions can be detected usingindirect photometric detection. Indirect photometric detection reliesupon the presence of a light absorbing electrolyte ion in the backgroundelectrolyte. Non-absorbing species are detected as zones of decreasedabsorbance or voids in the background due to the displacement of thelight absorbing electrolyte ion. Indirect photometric detection has beendescribed using fluorescent, ultraviolet (UV) and UV-visible (UV-vis)absorbing ions in the background electrolyte. For example, Small et al.in U.S. Pat. No. 4,414,842 describe a technique for detecting ions in anion exchange chromatography system by indirect UV detection in which aUV-absorbing ion is included in the elution buffer. Methods utilizingindirect photometric detection in capillary electrophoresis have beendescribed by Foret et al., J. Chromatography, 470:299-308 (1989); Kuhret al., Anal. Chem., 60:2642-2646 (1988); Kuhr et al., Anal. Chem.,60:1832-1834. However, these and other methods have not provedsatisfactory. For example none of these methods were able to separateand detect a mixture of eight standard anions (Br⁻, Cl⁻, SO₄ ⁻, NO₂,NO₃, F⁻, HPO₄ ⁻ and CO₃ ⁻). The main reason is the inability ofpreviously reported indirect photometric methods to provide the samelevel of sensitivity for UV transparent ions (e.g., F⁻, Cl.sup. -, SO₄⁻) and UV-absorbing ions (e.g., NO₂ ⁻, NO₃ ⁻). All published CE methodshave failed to successfully separate ions of widely differingproperties. e.g., slow migrators such as F⁻, PO⁻ and fast migrators suchas Br⁻, SO₄ ⁻. The need exists for a method for separating and detectingthese and other ionic molecules which is faster, more efficient, hasbetter resolution and requires less sample preparation than theavailable methods.

SUMMARY OF THE INVENTION

The present invention relates to methods for separating and detectingions by CE using carrier electrolyte solutions which facilitatedetection by indirect methods, particularly UV/visible spectroscopy. Thepresent methods rely upon reagents which can simultaneously effect asensitive, high resolution separation of several widely different ionicspecies, ranging from simple inorganic ions to complex organic ions, andboth slowly migrating and quickly migrating ions. Methods for separatingboth anions and cations are disclosed.

The methods generally involve introducing a sample containing the ionsinto a CE system which utilizes reagents which provide a light-absorbingbackground at a wavelength suitable for sensitive and interference-freeindirect photometric detection of all ionic species without regard totheir respective intrinsic UV absorption properties.

The sample is injected into a capillary filled with the carrierelectrolyte containing the reagent mixture, an electric current isapplied to the capillary under conditions appropriate to cause the ionsin the mixture to move toward the oppositely charged electrode and theionic species are detected photometrically.

The reagent mixture which is most effective as a component in a carrierelectrolyte for separating anions consists of the salt of a UV-absorbinganion (e.g., iodide, tungstate, molybdate, chromate, ferrocyanide,ferricyanide or vanadate). Chromate and vanadate compounds are preferredreagents for most anion separations, in part because of their ionicmobilities relative to the common inorganic anions and because of theirunusually broad UV spectra. In addition, one or more reagents forcontrolling the speed and/or direction of the electroosmotic flow of thecarrier electrolyte can, optionally, be included in the electrolytemixture. For example, an alkyl quaternary ammonium, phosphonium orarsonium salt having at least eight carbon atoms in a linear or branchedconfiguration can be added. Sodium chromate is a particularly preferredUV-absorbing salt and tetradecyltrimethylammonium bromide (TTAB) orcetyltrimethylammonium bromide (CTAB) are particularly preferred flowmodifiers. Alternatively, the carrier electrolyte can contain only thesalt of a UV-absorbing anion while the ammonium, arsonium or phosphonismgroups are bound (chemically or by absorptive forces) to the capillarywall.

In addition to the UV-absorbing anion and the flow modifier, anelectromigrative agent can be added to the system. The electromigrativeagent which enhances the detection of trace anions, e.g., speciespresent in nanomole concentrations and is generally added to the samplecontaining the analyte ions.

A reagent composition which is an effective carrier electrolyte forseparating cations is also the subject of the present invention. Thereagent is selected to allow separation and detection of cations havingwidely different properties (e.g., alkali and alkaline earth cations ina mixture with transition metals). This reagent composition consists ofa UV absorbing amine, such as 4-methylbenzylamine, heterocycliccompounds with or without sulfonic groups, such as, for example,2[N-morpholino] ethanesulfonic acid (MES) or naphthalene sulfonic acid,alkyl or aryl sulfonic acids, with or without additional UV absorbinggroups, such as, for example, dodecylsulfonic acid. The carrierelectrolyte can, optionally, also contain one or more chelating orcomplexing agents. The chelating or complexing agents are particularlyuseful for separating cations having the same or very close mobility.

The chemistry necessary to perform CE separations of ionic species forindirect detection can be contained in a kit. Such a kit for separatinganions would contain, inter alia, one or more light absorbing ionsspecific for the UV/visible range, such as a chromate and/or vanadatesalt, and optionally, a quaternary ammonium, arsonium or phosphoniumcompound. For detecting cations, the kit would contain a UV absorbingcation, such as 4-methylbenzylamine or MES, and optionally, one or morecomplexing or chelating agents, which are added to the sample in caseswhere groups of cations having approximately the same mobilities must beseparated.

The present reagent compositions and methods have several advantages,such as improved sensitivity, linearity of the range of calibration, theability to separate and resolve a wide range of anionic and cationicspecies, the ability to detect ionic species which are not detectable bydirect methods in addition to ions that are detectable by directmethods, less sample preparation and faster separation. The methods canbe used to separate and detect both simple and complex anions orcations, and to detect a variety of analytes simultaneously.

DESCRIPTION OF THE FIGURES

FIG. 1 is a chromatogram showing the separation of nineteen anions by CEusing TTAB/Na₂ CrO₄ as the carrier electrolyte.

FIG. 2 compares (A) a chromatogram showing the separation of fifteenanions using IC; with (B) a chromatogram showing separation of the samemixture of anions by CE using TTAB/Na₂ CrO₄ as the carrier electrolyte.

FIG. 3 is a chromatogram showing the separation of nine anions by CEusing Na₂ CrO₄ as the carrier electrolyte and modifying the capillarywalls with an uncharged polymer, effectively shielding the silanolgroups on the walls.

FIG. 4 is a chromatogram showing the separation of five anions by CEusing Na₂ CrO₄ and an amphoteric flow modifier as the carrierelectrolyte.

FIG. 5 is a chromatogram showing the separation of eight anions by CEusing TTAB/Na₂ CrO₄ and octanesulfonate as an electromigrative agent.

FIG. 6 is a chromatogram showing the separation of thirty anions by CEusing TTAB/Na₂ CrO₄ and electromigrative sample injection.

FIG. 7 is a chromatogram showing the separation of nine anions by CEusing naphthalene sulfonate as the electrolyte.

FIG. 8 is a chromatogram showing the separation of seven cations by CEusing 4-methylbenzylamine as the carrier electrolyte.

DETAILED DESCRIPTION OF THE INVENTION

The present method utilizes CE to simultaneously separate and detectionic species having widely different properties contained in a sampleusing indirect UV/visible detection. Indirect UV/visible spectroscopy isused because many ionic species cannot be detected using directdetection methods. CE is a well known technology, and has been describedin detail, for example, by Compton and Brownlee in Biotechniques,6(5):432-440 (1988); and Jorgenson and Lukacs in Science, 222:266-272(1983). A method of utilizing indirect photometric detection in CE isdescribed by Foret et al. in J. Chromatography, 470:299-308 (1989).

The present method is generally carried out using the followingprocedure: a capillary tube is filled with an electrically conductiveliquid (the carrier electrolyte) containing one or more reagents whichfacilitate detection by UV/visible spectroscopy. A preferred capillaryis generally a fused silica capillary having an internal diameter offrom about 50 to 100 microns (μ).

The ionic sample is introduced into the capillary, for example, byhydrostatic pressure, vacuum or by electromigrative injection in whichthe liquid sample is moved into the capillary by an electric current.After introduction of the sample, each end of the capillary is immersedin a reservoir which contains an electrode and the carrier electrolytesolution containing the reagents. The capillary tube is positioned witha detector on the column near the end opposite to sample introduction.Electric current is applied causing the anions or cations to move alongthe capillary toward the opposite electrode. The ions move at differentspeeds, depending upon several factors, such as their size and mobility.The electrophoretic separation is preferably monitored by indirectUV/visible spectroscopy. Other indirect detection methods can be used,however, UV/visible spectroscopy is preferred because it allowssensitive rapid detection of ionic species and is less costly than laserenhanced fluorescence detection, for example.

The method relies upon reagents which facilitate detection by indirectUV/visible spectroscopy, comprising light-absorbing compounds specificfor the UV/visible range. For detecting anions, a UV-absorbing anion isused and for detecting cations, a UV-absorbing cation is used in thecarrier electrolyte.

UV/visible light-absorbing compounds which are useful for separating anddetecting anions are UV-absorbing anions, such as iodide, tungstate,molybdate, chromate, ferrocyanide, ferrocyanate and vanadate salts.Absorbing anions which are particularly useful are selected chromate andvanadate salts. A preferred chromate salt is sodium chromate (Na₂ CrO₄)having a concentration of from about 1 mM to about 20 mM. A preferredvanadate salt is sodium vanadate having a concentration of from about 1mM to about 10 mM.

The carrier electrolyte can also contain, in addition to theUV-absorbing anion, a flow modifier, which is a compound which slows,stops or reverses the electroosmotic flow of the carrier electrolyte.Electroosmotic flow is the bulk flow of the electrolyte through acapillary that is induced by an applied electric field. The amount offlow and its direction is dependent on the charge of the inner wall ofthe capillary. If there is no wall charge, there is no electroosmoticflow. Thus, flow modifier can eliminate or reverse the effects of thecapillary wall on the flow of the electrolyte. Negating or counteractingthe wall effects can improve the resolution of the desired analyte ions.Flow modifiers which are useful in the present method include cationicsurface active agents, such as alkyl ammonium, arsonium and phosphoniumcompounds containing at least eight carbon atoms in a linear or branchedconfiguration. Such compounds include for example, quaternary ammoniumsalts, arsonium salts and phosphonium salts, biammonium salts,biphosphonium salts and biarsonium salts. These compounds include, forexample, octyl trimethylammonium, phosphonium or arsonium, various alkylderivatives of 1,8-diaminooctane, 1,8-diphosphinooctane or1,8-diarsinooctane. Also suitable are some polymeric ammonium,phosphonium and arsonium salts, such as, for example, hexadimethinebromide. Amphoteric ammonium compounds such as, for example3(N,N-dimethylpalmityl-ammonio)propanesulfonate are also useful flowmodifiers. Compounds which are particularly useful are quaternaryammonium salts which contain alkyl groups having at least eight carbonatoms in a linear or branched configuration. Preferred quaternaryammonium salts are tetradecyltrimethylammonium bromide (TTAB) and/orcetyltrimethylammonium bromide (CTAB). A concentration of TTAB or CTABof from about 0.1 mM to about 5.0 mM is useful in the present method.The use of flow modifiers facilitates control of both the direction, aswell as the rate of electroosmotic flow. Control of this parameterpermits the development of an assay that is both high in resolution andis complete in a short period of time. The carrier electrolyte solutiongenerally has a pH of from about 7.5 to about 8.5. An acid, such assulfuric acid or chromic acid, can be added to the electrolyte solutionto adjust the pH to the desired level.

In another embodiment of the present method, various aromatic carboxylicacids can be used as components in carrier electrolytes. The mainusefulness of carboxylates as carrier electrolytes is in the CE analysisof less mobile anions (e.g., fluoride, carboxylic acids,alkylsulfonates), which may produce broadly asymmetric peaks if analyzedusing the chromate electrolyte. However, because of their relatively lowmobilities, aromatic carboxylates are less suitable than chromates aselectrolytes for analysis of complex mixtures of highly mobile inorganicanions. The best results are obtained in CE when the mobilities betweenthe main anionic components of the carrier electrolyte and the analyteions is closely matched. Therefore, a range of highly UV-absorbingcarrier electrolytes covering the range of ionic mobilities of allinorganic anions and other low molecular weight species is of practicalinterest. Aromatic carboxylates are useful in the present method fordetecting and measuring some organic anionic species having low ionicmobility and which are UV-transparent, such as carboxylic acids, aminoacids, carbohydrates or sulfonates. Aromatic carboxylates such asphthalate, trimesate, benzenetetracarboxylate, p-hydroxybenzoate, andp-anisate are useful for this purpose.

In another embodiment of the present composition and method forseparating and detecting anions, an electromigrative agent can be addedto the sample in order to enhance the separation and/or detection oftrace amounts of anions, e.g. nanomolar quantities. The addition of anelectromigrative agent to the sample provides enrichment of theseparation of anions present in the sample in concentrations in thenanomolar range. In this embodiment, the agent is added to the sample,and the sample is injected into the capillary using electromigrativesample introduction. Electromigrative sample introduction involvesapplying a current having very low amperage which selectively causes thetrace anions to migrate toward the capillary. The addition to the sampleof the electromigrative agent, which has a lower ionic mobility incomparison to the carrier electrolyte anion, results in the selectivemigration of the trace anions into the capillary, which effectivelypreconcentrates these anions, thereby enriching the sample to beanalyzed with the trace anions. In addition to the analyte anions, it isalso possible to observe the enrichment of sample matrix anions actingas an isotachophoretic terminating electrolyte. Such anions may be addedpurposely. In solutions containing total ionic concentrations in thenanomolar range, sample conductivity often becomes too low, and can beadjusted by a suitable additive to enable sufficient electric chargethroughput for ionic transfer from the bulk of the sample solution intothe capillary. For this purpose, citrate, carbonate and octanesulfonatesalts can be used as electromigration additives.

Citrate, carbonate and octanesulfonate salts which exhibit lower ionicmobilities in comparison with the UV-absorbing anion in the carrierelectrolyte (e.g., chromate) can be used as additives forelectromigrative trace enrichment with the UV-absorbing anions in thecarrier electrolyte. Sodium octanesulfonate adjusted within the range of15 to 40 μm is particularly useful electromigration agent in low ioniccontent samples. Addition of relatively excessive concentrations ofoctanesulfonate does not lead to interfering comigration with any ofover fifty anionic species analyzed by the present CE method. Sodiumoctanesulfonate can be obtained free of common ionic impurities whichcould disturb the quantitation of common anions such as sulfate andchloride in unknown samples. An example of a separation of commoninorganic anions at low ppb levels using sodium octanesulfonate as anelectromigrative agent is shown in FIG. 5. As indicated, the detectionlimits (calculated as noise times three in concentration units) for thisseparation are in the low nanomolar range. This represents at least ahundredfold increase in sensitivity in comparison with the resultsachievable in the same carrier electrolyte and with hydrodynamic sampleintroduction.

UV/visible light-absorbing compounds which are useful in the presentmethod for separating and detecting cations are UV-absorbing cations,such as 4-methylbenzylamine, 2-aminopyridine,2-amino-4,6-dimethylpyridine, MES,3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) andN-[2-hydroxyethyl]piperazine-N'-[3-propane sulfonic acid](EPPS).4-Methylbenzylamine is the preferred UV-absorbing cation in the presentmethod. The concentration of 4-methylbenzylamine is generally from about3 mM to about 10 mM. The reagent mixture for cation separation canoptionally contain a complexing or chelating agent, such asethylenediaminetetraacetic acid (EDTA), citrate, tartrate,hydroxyisobutyrate, oxalate and succinate. The complexing agent allowscations having the same or similar mobilities to be differentiated.

The present methods can be utilized to analyze most types of ionicspecies. Samples containing complex mixtures of ions, including anions,cations and organic compounds, for example can be analyzed using themethod. When a sample containing such a complex mixture is separatedusing the present methods and electrolyte carriers for separatinganions, for example, the detector is placed just before the anodeimmersed in an electrolyte and the cathode is placed in another portionof the same electrolyte at the opposite end. Thus, the cations in thesample will move away from the detector, and the organic species willmove very slowly toward the anode, thereby creating a window for theanionic species toward the detector. The anions move most rapidly towardthe detector, thus are most efficiently resolved. Where a method andelectrolyte carrier appropriate for separating cations is used, thepolarity of the electrodes is reversed, and the cations will move towardthe detector (i.e., toward the cathode) while the anions in the samplewill move away from the detector.

The present methods are useful for analyzing samples containing multipleionic species in the shortest time possible, or to scan an unknownsample for ionic compounds, since the methods and reagent mixtures canefficiently separate and resolve such mixtures. Samples which can beanalyzed using the present methods include water, foods, such as juices,biological fluids or industrial chemical mixtures.

In one embodiment of the present method, a sample containing eightcommon inorganic anions: bromide, chloride, nitrate, nitrite, sulfate,fluoride, phosphate and carbonate, was analyzed by CE using a mixture of0.5 mM TTAB and 5 mM sodium chromate (Na₂ CrO₄) having a pH of 8 as thecarrier electrolyte. All eight anions were detected by monitoring theabsorbance of the carrier electrolyte at 254 or 272 nm. Separation ofall eight anions was completed in about three minutes. The ionic specieswere separated based on their ionic mobilities. This is importantbecause the elution sequence using the present method is predictablebased on the known ion mobilities of various ions. This means that thechemical identity of an unknown analyte can be reliably determined fromits position in the elution order.

In another embodiment of the present method, a sample containing seveninorganic cations: potassium, barium, strontium, sodium, calcium,magnesium and lithium, was analyzed by CE using 5 mM 4-methylbenzylaminein 0.21 mM citrate having a pH of 5.5 as a carrier electrolyte. Allseven cations were detected by monitoring the absorbance of the carrierelectrolyte at 214 mM. Separation of all seven cations was complete inless than three minutes.

Separation of ionic species using the present compositions and methodsis superior to ion chromatographic separations of similar mixtures inseveral respects: improved separation efficiency, shorter runtime,better selectivity, linearity of the plot and improved sensitivity. Forexample, the number of theoretical plates for sulfate in theillustrative example used above is 157.344. The highest plate-countsattainable by ion chromatography are smaller than 10,000. Separation ofthe standard eight anions was completed in three minutes by the presentmethod, whereas ion chromatographic separations of identical mixturestake typically six to fifteen minutes. Injection volumes for the CEseparation are less than about 40 nanoliters (nl) compared to about 50to 100 microliters (μl) for IC. Even though only 20 nL were injected toobtain the above separation, detection limits for all separated anionswere either comparable or better than those observed in IC. Thiscorresponds to a 10,000 fold increase in absolute sensitivity (per μginjected) in the present CE system in comparison with IC.

The present methods provide ionic separations which are efficient,highly selective, and which have a predictable order of elution. Themethods exhibit increased selectivity for ionic separations as comparedto other methods such as IC. During a typical CE separation of anionsusing the present chromate reagent mixture, cationic compounds migratein the opposite direction away from the anions of interest and are notseen in the electropherogram. Conversely, during a typical CE separationof cations using the methylbenzylamine reagent mixture, anioniccompounds migrate in the opposite direction away from the cations ofinterest. Neutral and slightly polar impurities are considerably lessmobile than the anions or cations and have longer migration times. Thus,the anions or cations of interest are efficiently separated and resolvedin the shortest time.

The practical usefulness of such increased selectivity can beillustrated, for example, using a fruit juice as the sample. When orangejuice is directly injected into an IC system, the first five peaks toelute, which represent fluoride, chloride, nitrite, bromide and nitrateions, are subjected to interference by carboxylic acids, such as formateor acetate and other organic compounds in the sample. To reduce thisinterference, analysis of the anions in the juice using IC would requirea complicated pretreatment of the sample to remove the carboxylates andorganic compounds. The same sample can be successfully analyzed by CE,and good separation of the anions can be obtained without anypretreatment of the sample using the present method.

The invention is further illustrated by the following Examples.

EXAMPLE 1 General Procedure for CZE of Anions Using Na₂ CrO₄ /TTABElectrolyte

A sample containing the following eight inorganic anions was prepared:fluoride (F). carbonate (CO₃), chloride (Cl), nitrite (NO₂), nitrate(NO₃), bromide (Br), phosphate (H₂ PO₄) and sulfate (SO₂).

A fused silica capillary externally coated with polyimide (PolymicroTechnologies) was freshly cut from a roll and approximately 1 cm sectionof polyimide coating was burned off with a butane lighter for UV to passthrough at 40.5 cm from one end. The total capillary length was 63 cm.and had an internal diameter of 75 μm. The capillary was installed intothe cell and purged with electrolyte with a 1 cc luer syringe with anadapter. The electrolyte was 5 mM Na₂ CrO₄ and 0.5 mM TTAB, adjusted topH 8 with 10 mN sulfuric acid. A 50 ml beaker and a 100 ml beaker werefilled with electrolyte to equal heights. The 50 ml beaker was placed atthe cathode end of the capillary and the 100 ml was placed at the anodeend Approximately 100 microliters of carrier electrolyte was run throughthe capillary prior to analysis.

The power supply (Spellman (0 to 30 KV)) was manually turned to zero.The capillary at the cathode end was picked up manually, raised to 16 cmheight above the electrolyte level and placed in the sample for 30seconds. The capillary was removed from the sample and placed promptlyinto the electrolyte. The voltage was manually ramped from 0 to 20 KVduring approximately 10 seconds while the start integrate signal wasinitiated at the beginning of the voltage ramp. At 20 KV a typicalcurrent reading was about 20 μA.

Detection was carried out using a Linear Instruments variable UV/Vis CEdetector at two different wavelengths: 254 nm and 272 nm.

Separation was completed in about three minutes, and a clear anddistinct peak was obtained for each anion. All eight anions wereseparated within about one minute.

EXAMPLE 2 CE Separation of a Complex Mixture of Anions

The separation of a complex mixture of ten (10) weakly and stronglydissociated anionic species was carried out according to the proceduredescribed in Example 1. The ten anions in the mixture were: Cl, SO₄,NO₃, F, CO₃, formate, acetate, propionate, butyrate and an unidentifiedorganic acid. Separation was completed within about 3.8 minutes. All tenanions eluted and were detected, and a clear and distinct peak wasobtained for each anion.

EXAMPLE 3 CE Separation of a Complex Mixture of Nineteen Anions

The separation of a complex mixture of nineteen anionic species wascarried out according to the general procedure described in Example 1.The injection volume was 20 nl, and indirect UV/visible detection wascarried out at 272 nm. The nineteen anions were:

    ______________________________________                                                   anion      ppm                                                     ______________________________________                                        1.               bromide      4                                               2.               chloride     2                                               3.               sulfate      4                                               4.               nitrite      4                                               5.               nitrate      4                                               6.               molybdate    20                                              7.               citrate      4                                               8.               fluoride     1                                               9.               phosphate    4                                               10.              phoshite     4                                               11.              phthalate    4                                               12.              methanesulfonate                                                                           5                                               13.              ethane sulfonate                                                                           5                                               14.              acetate      5                                               15.              propanesulfonate                                                                           5                                               16.              butane sulfonate                                                                           5                                               17.              benzoate     5                                               18.              pentane sulfonate                                                                          5                                               19.              hexane sulfonate                                                                           5                                               ______________________________________                                    

Separation was completed in less than four minutes. All nineteen anionswere detected and a clear and distinct peak was obtained for each anion,as shown in FIG. 1. The numbers on the peaks correspond to the numbersin the above list of anions.

EXAMPLE 4 Comparison of a CE Separation and an IC Separation of FifteenAnions

The separation of a mixture of 15 anions was carried out by CE accordingto the procedure set out in Example 3. The same mixture was separated byIC according to standard IC conditions. The fifteen anions were:

    ______________________________________                                                  anion        ppm                                                    ______________________________________                                        1.              thiosulfate    4                                              2.              bromide        2                                              3.              chloride       2                                              4.              sulfate        4                                              5.              nitrite        4                                              6.              nitrate        4                                              7.              molybdate      20                                             8.              tungstate      20                                             9.              monofluorophosphate                                                                          4                                              10.             citrate        4                                              11.             fluoride       1                                              12.             phosphate      4                                              13.             phosphite      4                                              14.             phthalate      4                                              15.             carbonate      4                                              ______________________________________                                    

The results are shown in FIG. 2. FIG. 2A is a chromatogram of the ICresults after 2.5 minutes. The large rounded peak represents thecarbonate ion (HCO₃ ⁻), and the curve which starts upward at about the2.5 minute mark represents the start of the chloride ion (Cl⁻) peak.

FIG. 2B is a chromatogram showing the CE separation. Separation of allfifteen anions was completed in about 2.5 minutes, and a clear anddistinct peak was obtained for each anion.

The results showed that for identical ppm levels of each anion,approximately the same signal to noise ratios were observed by CE froman injection volume of 20 nL as by IC for an injection volume of 100 μl.These results indicate that the CE method is about 5000 times moresensitive than conventional IC. In this example, it took about twominutes for an average IC peak to elute under standard conditionswherein the CE method separated fifteen peaks in the same period oftime. The observed increase in sensitivity is due to increasedseparation efficiency: about 1000 theoretical plates for IC vs. about100,000 for CZE.

EXAMPLE 5 CE Separation of a Mixture of Nine Anions Using a ModifiedCapillary

The separation of a mixture of nine anions was carried out according tothe procedure described in Example 1. except that no flow modifier(TTAB) was used. The capillary wall was modified by covering the innerwall with a layer of PSDVB polymer to shield the negative charges of thesilanol groups present on the wall. The capillary was 46 cm in lengthand had an internal diameter of 50 μm. All nine anions were separated bythe procedure in less than nine minutes, as shown in FIG. 3. The anionsshown in FIG. 3 are:

1. thiosulfate

2. bromide

3. chloride

4. sulfate

5 nitrite

6. nitrate

7. molybdate

8. azide

9. tungstate.

EXAMPLE 6 CE Separation of Five Anions Using an Amphoteric Flow Modifier

Separation of a mixture of five anions was carried out according to theprocedure described in Example 1, except that an amphoteric detergent,3(N,N-dimethylpalmitylammonio)propanesulfonate (pH 8, 0.5 mM), was usedin lieu of TTAB. The capillary was 60 cm in length and had an internaldiameter of 75 μm. All five anions were separated in about nine minutes,as shown in FIG. 4. The anions shown in FIG. 4 are:

1. Br

2. Cl

3. SO₄

4. NO₂

5. NO₃

EXAMPLE 7 Improving Sensitivity of Separation of an Eight Anion Mixtureby Electromigrative Sample Introduction

CE separation of an eight anion mixture was carried out as described inExample 1 above, except that an electromigration enhancer sodiumoctanesulfonate, was added to enhance sensitivity for trace amounts ofanions.

The carrier electrolyte contained 5 mM chromate and 0.5 mM TTABelectroosmotic flow modifier and was adjusted to pH 8.1. Fused silicacapillary (75 μm internal diameter 52 cm from the point of sampleintroduction to the detector) was used for the separation. During theanalysis, the injection side was at -20 kV. The electromigrative sampleintroduction was carried out at 5 kV for 45 seconds. Sample conductivitywas adjusted by the addition of sodium octanesulfonate at 18 μN to thesample. The peak identities, ppb concentrations and nM detection limits(3× the noise), shown in FIG. 5, were as follows: Peak 1:Bromide 4 ppb,13.6 nM; 2:Chloride 4 ppb, 13 nM; 3:Sulfate 4 ppb, 8.4 nM, 4:Nitrite 4ppb, 25.4 nM; 5:Nitrate 4 ppb, 24 nM; 6:Fluoride 2 ppb, 19.8 nM and7-Phosphate 8 ppbm 17.8 nM. The large peak at about 3.2 minutes is thecarbonate. The levels of carbonate were not controlled under theconditions of this experiment. These results show that the detectionlimits (calculated as 3× noise in concentration units) for thisseparation are in the two nanomolar range, which represents at least ahundredfold increase in sensitivity in comparison with the resultsachievable in the same carrier electrolyte without the addition ofsodium octanesulfonate to the sample.

EXAMPLE 8 CE Separation of a Thirty anion Mixture Using ElectromigrativeSample Introduction

CE separation of a thirty anion mixture was carried out as described inExample 7, using electromigrative sample introduction. Theelectromigrative sample introduction was carried out at 1 KV for 14seconds. The capillary was 60 cm in length and had an internal diameterof 50 μm. The electrolyte was 5 mM Na₂ CrO₄ and 0.5 mM TTAB, pH 8.0. Allthirty anions were separated in less than three minutes, as shown inFIG. 6. The anions shown in FIG. 6 are listed below:

1. thiosulfate

2. bromide

3. chloride

4. sulfate

5. nitrite

6. nitrate

7. molybdate

8. azide

9. tungstate

10. monofluorophosphate

11. chlorate

12. citrate

13. fluoride

14. formate

15. phosphate

16. phosphite

17. chlorite

18. galactarate

19. carbonate

20. acetate

21. ethanesulfonate

22. propionate

23. propanesulfonate

24. butyrate

25. butanesulfonate

26. valerate

27. benzoate

28. glutamate

29. pentanesulfonate

30. gluconate

EXAMPLE 9 CE Separation of Nine Anions Using Naphthalene Sulfonic AcidElectrolyte

A mixture of nine anions was separated according to the proceduredescribed in Example 1. The nine anions, designated C4-C10, C12 and C14,are linear alkylsulfonates having from 4 to 14 carbon atoms, at aconcentration of 25 ppm each. A capillary 60 cm long and having aninternal diameter of 75 μm was used. The electrolyte was 10 mMnaphthalene sulfonic acid (30% ACN) adjusted to pH 10 with NaOH. Thesample was injected by hydrostatic injection for 30 seconds. Indirect UVdetection was used at a wavelength of 254 nm.

The results are shown in FIG. 7. All nine anions were separated andelution was complete in about fifteen minutes.

EXAMPLE 10 General Procedure for CE Separation of Cations Using4-Methylbenzylamine Electrolyte

A sample containing the following seven inorganic cations was prepared:potassium (K), barium (Ba), strontium (Sr), sodium (Na), calcium (Ca),magnesium (Mg) and lithium (Li).

A fused silica capillary externally coated with polyimide was freshlycut from a roll and prepared as described in Example 1. The totalcapillary length was about 60 cm, and had an internal diameter of 75 μm.The electrolyte was 5 mM 4-methylbenzylamine and 0.021 mM citrate, pH5.5. The pH was adjusted to pH 5.5 using 2N morpholinoethanesulfonate(MES). A 50 ml beaker and a 100 ml beaker were filled with electrolyteto equal heights. The 50 ml beaker was placed at the anode end ofcapillary and the 100 ml beaker was placed at the cathode end.

The sample was injected by hydrostatic injection and the separationcarried out according to the procedure described in Example 1. Thevoltage was 25.0 KV Detection was carried out using a UV/Vis CE detectorat a wavelength of 214 nm.

The results are shown in FIG. 8. All seven cations were separated inless than three minutes, and a clear and distinct peak was obtained foreach cation. All seven cations were separated within about one minute.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

We claim:
 1. A method for detecting anions in a sample using capillaryzone electrophoresis comprising the steps of:a. introducing the sampleinto a capillary; b. immersing the capillary in a carrier electrolytecontaining a chromate salt; c. applying an electrical current underconditions appropriate for the anions in the sample to move along thecapillary toward the anode thereby causing separation of the anions tooccur; and d. detecting the anions indirectly using a UV/visiblephotometric detector.
 2. The method of claim 1 wherein the chromate saltis sodium chromate having a concentration of from about 1.0 mM to about20 mM.
 3. The method of claim 1 wherein an alkyl quaternary ammoniumsalt, arsonium salt or phosphonium salt containing at least one alkylgroup having more than eight carbon atoms is added to the carrierelectrolyte.
 4. The method of claim 3 wherein the alkyl quaternary saltis tetradecyltrimethylammonium bromide having a concentration of fromabout 0.1 mM to about 5.0 mM.
 5. The method of claim 3 wherein the alkylquaternary salt is cetyltrimethyl ammonium bromide having aconcentration of from about 0.1 mM to about 5.0 mM.
 6. The method ofclaim 1 wherein the alkyl quaternary ammonium, arsonium or phosphoniumsalt is immobilized on the capillary wall.
 7. The method of claim 1wherein electrical voltage is from about 5 to about 40 KV.
 8. The methodof claim 1 wherein an electromigration additive is added to the sample.9. The method of claim 8 wherein the electromigration additive isselected from the group of: an octanesulfonate salt, a carbonate saltand a citrate salt.
 10. The method of claim 9 wherein theelectromigration additive is sodium octanesulfonate.
 11. The method ofclaim 1 wherein the anions are complex organic anions selected from thegroup consisting of: anionic complexes of metals, carboxylic acids,sulfonic acids and alkyl sulfates.
 12. The method of claim 1 wherein theanions are inorganic anions.
 13. The method of claim 1 wherein avanadate salt is used in lieu of the chromate salt.